Various types of cancers occur throughout the body, and affect large numbers of people. It is postulated that many of these cancers are caused by foreign substances, also referred to as xenobiotics. One method of limiting the carcinogenic effect of various xenobiotics is by promoting steps or increasing levels of substances in the metabolic pathway that allow carcinogens to be metabolized into neutral forms that are easily excreted from the body.
A simplified description of how xenobiotics, compounds foreign to the body, are metabolized is that procarcinogens are metabolized by Phase I enzymes to either (i) electrophilic metabolites, which may cause DNA damage leading to DNA repair mechanisms or to cancer, or to (ii) nonelectrophilic metabolites that are further metabolized by Phase II enzymes to produce detoxification products. Several steps in this pathway potentially neutralize xenobiotics, and therefore could be considered as logical targets for chemoprevention of cancer. For example, the induction or inhibition of phase I enzymes might promote neutralizing metabolic steps, as could the induction of phase II enzymes. Alternatively, the promotion of DNA repair is a potentially promising means of preventing the carcinogenic effect of xenobiotics that are metabolized through Phase I enzymes to produce electrophilic metabolites which damage nucleic acid structures.
The prevention of the development of cancer by administering drugs prophylactically has been termed chemoprevention. Chemoprevention is an emerging concept that envisages the active prevention of malignant processes. Chemoprevention involves the "use of specific natural or synthetic substances with the objective of reversing, suppressing or preventing carcinogenic progression . . . " (Singh and Lippman, 1998). A number of such chemopreventive agents are currently in development for undergoing clinical trials.
Chemopreventive agents can be conceptually classified as "blocking" or "suppressive" agents. Blocking agents prevent cancer-producing compounds from reaching or reacting with their critical target sites; suppressive agents prevent the evolution of the neoplastic process in cells already altered by carcinogens (see Singh and Lippman, 1998 for review). Thus, chemopreventive agents can be provided either to high risk groups or to the population at large.
Oltipraz can be considered to have both blocking and suppressive activities, and is being investigated for chemopreventive activity in a number of cancers, including those of the bladder, prostate, breast, skin, lung, colon, and liver (Wattenberg, 1997). A large scale clinical trial of the ability of oltipraz to prevent aflatoxin F1-related hepatocellular carcinoma is currently underway in the People's Republic of China. However, oltipraz has been associated with some toxicities, including photosensitivity/heat intolerance, GI effects, and neurologic toxicities (Dimitrov et al., 1992). Thus, the continued search for improved chemopreventive agents is clearly warranted.
Phase II enzymes are responsible for the detoxification of reactive electrophilic and nucleophilic metabolites. Phase II enzymes include NAD(PH):quinone oxidoreductase (facilitates metabolism of carcinogens through two electron reduction), glutathione S transferase (mediates deactivation of carcinogens through conjugation to reduced glutathione), manganese superoxide dismutase (reduces levels of superoxide anion) ferritin (reduces oxygen free radical formation by sequestering iron), and others (Talalay, 1989). Glutathione S-transferases (GST) are phase II enzymes that catalyze the reaction of glutathione, a tripeptide, with electrophiles such as epoxides, alkyl and aryl halides and .alpha.,.beta.-unsaturated ketones. Glutathione conjugation serves to deactivate electrophiles, therefore making them less toxic and carcinogenic and more easily excreted by the body.
In the reaction between glutathione and an electrophile, the first step involves the binding of glutathione (GSH) to the glutathione-S-transferase enzyme (GST). The enzyme is known to lower the pKa of the thiol from 9.0 to .about.6.5. The thiolate ion of glutathione then reacts with electrophiles to produce the less toxic glutathione conjugates.
Several sulfur-containing compounds are known to elevate levels of GST in rats and mice. These include allicin (Talalay, et al.), a natural product found in garlic, and Oltipraz 10, which is undergoing clinical trials at the time of the present disclosure.
Oltipraz and its derivatives (1,2-dithiole-3-thiones) are particularly attractive because of two important characteristics. First, 1,2-dithiole-3-thiones are monofunctional inducers. They only induce phase II enzymes and not phase I enzymes. Induction of phase I enzymes could enhance the production of activated carcinogens, therefore complicating any increased activity of phase II enzymes. Secondly, 1,2-dithiole-3-thiones can induce higher levels of GSTs in several organs, providing possible protection against several types of cancer. ##STR1##
Because of this selective usefulness of sulfur-containing compounds that display an ability to induce high levels of GST in particular, industry is constantly seeking additional forms of such compounds, as well as reliable methods for their synthesis. As a result, several syntheses have been reported for unsaturated five-membered cyclic sulfenate esters or .gamma.-sultines (Bondarenko, et al.); however, none of the reported syntheses to date are very general. In 1970, King and co-workers reported that thermolysis of thiete 1,1-dioxide and 2-phenylthiete 1,1-dioxide produced unsaturated sultines (King et al.) Thermolysis of other substituted thiete 1,1-dioxides resulted in sulfur dioxide extrusion to yield various alkenes.
Braverman and co-workers have studied the electrophilic fragmentation and cyclization of allenic sulfones to unsaturated .gamma.-sultines (Braverman and Duar). Allenyl sulfone when reacted with bromine produced a bromonium ion intermediate which cyclized to produce sultine. This reaction is unfavorably limited, however, because only bromine substituted sultines can be synthesized.
Another synthesis of unsaturated sultines reported by Duboudin and co-workers required Grignard reagents formed from propargyl alcohols (Thomazeau et al.). The resulting Grignards reacted with sulfur dioxide by insertion into the carbon-metal bond. The sultines were obtained, however, in poor to moderate yields.
Accordingly, novel compounds that can induce GST and other enzymes of the Phase II response, and a method of reliably synthesizing such compounds in various forms while resulting in useful levels of the desired product is still sought.
The tendency of HIV-1 to mutate to viral strains that are resistant to existing therapeutic regimens is now well documented (Cohen, 1997). This resistance has fueled the search for antiviral drug targets that are conserved through mutations. The zinc finger containing nucleocapsid proteins of retroviruses would appear to be such a therapeutic target since they are conserved through mutations, are involved in early and late phases of the viral replication cycle, and are chemically reactive toward soft electrophilic reagents that can be prepared via rational synthetic structure-activity modification schemes (Rice and Turpin, 1996). The structure of the HIV-1 nucleocapsid protein (NCp7) was determined in the early 1990's (South et al., 1990; Chance, et al., 1992; Summers et al., 1992) and found to have the Cys-Xaa.sub.2 -Cys-Xaa.sub.4 -His-Xaa.sub.4 -Cys zinc coordination sphere sequence with the short tether links that are now sometimes referred to as a "zinc knuckle". At that same time Rice and co-workers first postulated that the Cys residues of zinc fingers could be chemically modified by electrophilic attack. They first demonstrated the viability of this chemical postulate using 3-nitrosobenzamide (NOBA) (Rice et al., 1993). While NOBA reacted with the zinc fingers of NCp7 causing zinc ejection, it was reduced to an aromatic hydroxylamine. This class of compounds is known to be quite mutagenic (Nohmi, et al., 1984) so the Rice group initiated a search for other soft electrophilic functional groups which would react with the sulfur rich zinc finger domain.
The disulfide functional group has the desired chemical reactivity and the Rice group had access to a host of 2,2'-dithiobisbenzamides (DIBAs) (3) (submitted by Parke-Davis Pharmaceutical) via NCI's Developmental Therapeutics Program (Rice et al., 1995; Rice et al., 1996; Tummino et al., 1996). These compounds were used by Rice et al. (1995 and 1996) and the Parke-Davis group (Tummino et al., 1996) to definitively correlate ability to eject zinc from the NCp7 protein with the ability of the compounds to exert anti-HIV-1 activity. ##STR2##
Unfortunately, reaction of these acyclic disulfides (RSSR) (3) with intracellular reducing agents/nucleophiles such as reduced glutathione produced two thiols (RSH) which no longer react with the zinc fingers. This observation lead to a search for cyclic disulfides and analogs thereof, particularly for a molecule in which the 2 thiols could not dissociate from one another, thereby effectively rendering redox return to the disulfide impossible. Lamar Field's group had deposited a number of 1,2-dithiolanes (4) and 1,2-dithianes (5) in NCI's chemical repository and the Rice group screened a number of these (Rice et al., 1997). ##STR3##
Two of the six 1,2-dithiolanes (6, 7) and two (8, 9) of the 13 1,2-dithianes screened were active against HIV-1.sub.RF replication in CEM-SS cells in the XTT-based cytoprotection assay, were active against replication of monocytotropic HIV-1.sub.ADA in fresh human monocyte/macrophage cultures and promoted ejection of zinc from the NCp7 protein. Both the XTT and monocyte/macrophage assays were used because infection of monocyte/macrophage cultures is thought to resemble infection of the nonproliferating pool of cells in vivo, while the parameters for the CEM-SS cells (XTT assay) are thought to reflect more closely the parameters for highly proliferating cells such as bone marrow and intestinal cells. The CEM-SS cell (XTT) assay is a more sensitive indicator of toxicity and all compounds disclosed herein have been screened in this assay.
TABLE 1 Anti-HIV-1 and Zinc Ejection Screening of 6, 7, 8, 9 ##STR4## ##STR5## ##STR6## ##STR7## Monocyte/ XTT Macrophage Assay (.mu.M) Assay (.mu.M) # NSC # EC.sub.50 IC.sub.50 EC.sub.50 IC.sub.50 TSQ NCp7 Assay 6 661127 34 200 N/A* N/A* 7 661126 9.8 30 21 &gt;100 18 8 624151 6.6 184 8.0 &gt;100 17 9 624152 13 135 9.1 &gt;100 20 N/A* No material was available for further testing.
The most active, least toxic compound, NSC 624151 (8), was then screened for of antiviral action against a variety of strains of HIV-1 and found to be active (3-69 .mu.M depending on strain) against all tested strains. NSC 624151 (8) was found to promote zinc ejection from the NCp7 protein but have no other observable effect on enzymatic activities of the viral replication cycle.
The antiviral mechanism of action is postulated to be electrophilic attack of the oxidized organosulfur compounds on the nucleophilic/reducing cys residues present in the zinc fingers. Structurally related organosulfur compounds are known to be soft electrophiles and susceptible to nucleophilic attack by soft nucleophiles such as thiols and thiolate anions (Talalay et al., 1988). These types of compounds are also substrates for glutathione S-transferases, a family of enzymes involved in detoxification of electrophiles and chemoprevention of cancer (Wilce and Parker, 1994); Coles and Ketterer, 1990; Armstrong, 1991), and as discussed in the preceding paragraphs. Oltipraz (10) has also been shown to prevent HIV-1 replication (Prochaska et al., 1993). Since reduced glutathione is a potential competing intracellular nucleophile/reducing agent present in high concentrations in all cells, NSC 624151 (8) was also screened in the XTT cytoprotection assay in the presence of a two fold excess of glutathione (GSH). Antiviral activity was retained but the EC.sub.50 did increase by a factor of almost 10 from 6.6 .mu.M to 64.2 .mu.M. Toxicity was also decreased with GSH treatment with IC.sub.50 going from 184 .mu.M to &gt;200 .mu.M. The competing nucleophile/reducing agent glutathione does reduce but does not eliminate the anti-HIV activity of these compounds.
After discovery that the cyclic dithianes and dithiolanes still maintain anti-HIV-1 activity even in the presence of reduced glutathione, the Rice group began to tackle the critical question of zinc finger specificity. Can compounds be produced which will selectively target retroviral nucleocapsid protein zinc fingers without affecting other cellular zinc fingers? This group has now shown that dithiane (8) does not affect poly (ADP-ribose) polymerase (PARP) activity or alter specific binding of the zinc finger containing transcription factors Sp1 and GATA-1 to their DNA targets (Huang et al., 1998). Lastly, they also examined the effect of NSC 624151 (8) on in vitro transcription using HeLa nuclear extract. A CMV promoter was used to drive the transcription of a duck HBV sequence and 8 did not significantly alter this transcriptional process either.
Maynard and Covell have came out with a density functional theory (DFT) analysis, in combination with protein-ligand docking methods, for predicting reactivity of NCp7 with electrophilic reagents (Maynard et al., 1998). They found that the molecular property of these soft electrophiles that correlated most strongly with their reactivity toward NCp7 and zinc ejecting ability was the ratio of electronegativity to hardness (.chi..sup.2 /.eta.). This quantity is related to the capacity of an electrophile to promote a soft, covalent bond forming reaction. Calculation of .chi..sup.2 /.eta. for a variety of zinc ejectors was presented along with protein-ligand docking analysis.
From the DFT calculations, they observed a linear correlation between NCp7 reaction rates and the ligand's capacity to promote a soft, covalent reaction, .chi..sup.2 /.eta.. The most reactive DIBA compounds (3, R'=C(O)NH-phenyl-SO.sub.2 NH.sub.2 and R'=C(O)NH-phenyl-SO.sub.2 NHC(O)CH.sub.3 had .chi..sup.2 /.eta. values of 0.2935 and 0.3098 respectively. The next most reactive compound, dithiane, (9) had a .chi..sup.2 /.eta. value of 0.2260. Compounds with .chi..sup.2 /.eta. values of less than 0.2 exhibited relatively poor zinc ejecting ability.
The NMR structure of NCp7 was then used as a fixed model for ligand docking analysis. Zinc finger 1 and zinc finger 2 docks were examined and Fukui functions of the Cys thiolates (S.sup.15, S.sup.18, and S.sup.28 in finger 1 and S.sup.39 and S.sup.49 in finger 2) were calculated and their proximity to the ligand electrophilic sites in the best scoring docks were calculated. Zinc finger 2 is known to be 7 fold more reactive (Rice et al., 1995; Rice et al., 1996; Tummino et al., 1996; Huang et al., 1998) than zinc finger 1 with the DIBA compounds (3) and this observation correlated well with calculated binding proximities. Frontier molecular orbital Cys.sup.49 HOMO to electrophile LUMOs were then calculated and dithiane (9) showed high orbital overlap consistent with its chemical reactivity. This ligand docking analysis predicted a saddle shaped docking domain of each zinc finger and two nearly equivalent binding sites (designated .alpha. and .beta.) for most small molecules inside the saddle.
Rice, Turpin and co-workers have reported new compounds capable of binding to both the .alpha. and .beta. sites of zinc finger 2 and they included the Covell/Maynard calculations of their most likely binding conformations (Rice et al., 1999). In this study, they abandoned the disulfide linkage in 3 in favor of a thioester linkage (11). As mentioned earlier, acyclic disulfides can be deactivated in this chemistry by reduction to the thiolates. The monomeric thiolates can then simply diffuse away from one another rather than recombine after reoxidation. Cyclic compounds such as 9 do not suffer from this functional group disadvantage.
Extensive synthetic work reported in this paper resulted in the identification of pyridinoalkanoylthiolesters (PATEs) (11) as compounds that could span both a and .beta. docking sites and provide a soft electrophilic thioester group in close proximity to Cys.sup.49. These compounds had antiviral EC.sub.50 's in the XTT cytoprotection assay of 5-6 .mu.M and exhibited zinc finger reactivity, as measured by the Trp37 fluorescence decrease assay of 3-4 relative fluorescence units/min over 30 minutes. ##STR8##
The inventor is also familiar with work in which 32 different sulfur heterocycles and sulfur containing transition metal complexes were screened in the XTT cytoprotection assay and zinc ejection assay described herein above. Of the 18 metal complexes screened, only 1 (31) had antiviral activity and it did not have an EC.sub.50. Cytoprotection of 39% was observed at 100 .mu.M and its IC.sub.50 was 154.5 .mu.M. ##STR9##
It would be advantageous, therefore to provide specific antiviral agents, capable of ejecting zinc from viral zinc finger configurations, in order to effectively address the mutational defense mechanisms of HIV. The present disclosure is believed to address this deficiency in the art, by providing novel compounds with anti-HIV activity and that are also useful in the prevention of certain cancers.